Expression of the Aplysia californica rho Gene in Escherichia coli ...

Vol. 7, No. 10

MOLECULAR AND CELLULAR BIOLOGY, OCt. 1987, p. 3620-3628 0270-7306/87/103620-09$02.00/0

Expression of the Aplysia californica rho Gene in Escherichia coli: Purification and Characterization of Its Encoded p21 Product PAUL S. ANDERSON AND JUAN CARLOS LACAL* Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892 Received 28 May 1987/Accepted 22 July 1987

A nvw family of highly conserved genes, designated rho, has recently been isolated and characterized (P. Madaule and R. Axel, Cell 41:31-40, 1985). These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rats, and humans, and their 21,000-dalton products are highly homologous. The rho p21 protein shares 35% amino acid homology with the Harvey ras p21 protein and on this basis has been proposed to be a G protein. We expressed the Aplysia californica rho gene in Escherichia coli and purified its p21 protein to more than 90% purity. The availability of the rho protein in high quantities made it possible to establish its high affinity for guanine nucleotides. The rho p21 protein had nucleotide-binding properties similar to those of the ras p21 protein. However, a comparison of these proteins revealed some important differences regarding their specificities and affinities. Finally, the rho p21 protein had GTPase activity almost identical to that of a normal ras p21 protein, the rates being 0.106 and 0.105 mol/min per mol of p21, respectively. Thus, the results suggest that the degree of homology found between the ras and rho gene products most likely is related to the conservation of sequences relevant to their ability to bind and hydrolyze guanine nucleotides. The fact that the rho p21 protein binds and hydrolyzes GTP strongly suggests that it is a G protein with a potential regulatory function conserved in evolution.

Recently, the existence of a G protein responsible for hormones, neurotransmitters, and growth factor-dependent polyphosphoinositide breakdown has been postulated. Cleavage of inositol-containing phospholipids by phospholipase C produces inositol 1,4,5-trisphosphate and 1,2diacylglycerol. While inositol 1,4,5-trisphosphate acts as a second messenger mobilizing intracellular Ca2 , 1,2diacylglycerol activates protein kinase C. This putative G protein, Gp,, is sensitive to pertussis toxin only in some cell types (6), like the G0 of adenylate cyclase, but seems to be distinct from G1 in immunochemical analysis (10). Paradoxically, Gp is insensitive to pertussis toxin in other cell types

There is substantial evidence that G proteins are important regulatory elements in several cellular functions. One of the best characterized systems is the hormone-sensitive adenylate cyclase complex (11), consisting of several proteins, including two G proteins. The G, protein mediates the stimulation of adenylate cyclase activity, which is responsible for the production of cyclic AMP, by directly activating the catalytic subunit (11). The G, protein is responsible for its inhibition. Both Gs and Gi are active only when associated with GTP and become inactive by means of an intrinsic GTPase activity which renders the protein associated with GDP (11). The same cycle of activation and inactivation by specific association with GTP and GDP, respectively, is found in other systems. Transducin is responsible for the regulation of the levels of cyclic GMP through activation of a specific phosphodiesterase in the outer segment of the retinal rod by means of a light-inducible mechanism (30). A recently characterized G protein isolated from brain, G., seems to be responsible for the opening of Ca2" channels in neuronal cells (14). While it is well established that G0, Gi, Go, and transducin are composed of at least three subunits, a (39 to 45 kilodaltons [kDa]), P (35 kDa), and fy (-10 kDa), only the a subunit has the ability to bind GTP and GDP. G proteins play an important role in the initiation and elongation of protein synthesis as well. Initiation factor eIF-2 mediates the binding of the initiator Met-tRNA to the small ribosomal subunit via formation of a tertiary complex containing GTP (23). eIF-2 is composed of three subunits, eIF-2a (38 kDa), eIF-2p (-50 kDa), and eIF-2-y (-54 kDa), among which the a subunit probably binds guanine nucleotides. The bacterial elongation factors EF-Tu and EF-G and the eucaryotic elongation factor EF-1 belong as well to the family of G proteins and follow similar patterns of activation and inactivation by association with GTP or GDP. *

(6).

Another group of G proteins, designated generically as ras p21, are related to the regulation of cell proliferation and differentiation (7, 27, 31; J. C. Lacal and S. R. Tronick, in P. K. Reddy, T. Curram, and A. Skalka, ed., The Oncogene Handbook, in press). The still growing family of ras genes is highly conserved in evolution, with at least three members, Harvey ras, Kirsten ras, and N-ras, found in humans. Genes with a substantial degree of homology have been found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, Aplysia californica, Dictyostelium discoideum, and a variety of mammalian species (Lacal and Tronick, in press). Although not yet completely understood, the very similar proteins found in S. cerevisiae and in mammals seem to involve different functions (2). Recently, new families of genes have been identified on the basis of their homology to mammalian ras genes (1, 19, 20, 24). The rho gene was originally isolated from the mollusc A. californica and subsequently has been found in different species, including S. cerevisiae, D. melanogaster, rats, and humans (20). The products encoded by the mammalian Harvey ras and rho genes share 35% homology at the amino acid level. Thus, to establish whether the rho protein is a G protein, we expressed the A. californica rho gene in Escherichia coli. The strategy followed as well as the purification

Corresponding author. 3620

VOL. 7, 1987

EXPRESSION OF A. CALIFORNICA rho GENE IN E. COLI

and biochemical characterization of the rho protein are described. MATERIALS AND METHODS Plasmid constructions. Digestion of DNAs was performed in accordance with the instructions of the restriction enzyme supplier (New England BioLabs, Inc., Beverly, Mass.). Dephosphorylation of digested plasmids was carried out with calf intestinal alkaline phosphatase (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) in 10 mM Tris hydrochloride-10 mM MgCl2 (pH 8.5) for 1 h. Ligation of inserts to plasmids was performed in 60 mM Tris hydrochloride (pH 7.6)-10 mM MgCl2-10 mM dithiothreitol (DTT)-1 mM ATP-1 mM spermidine at 14°C for 14 h. Bacterial transformations were carried out as previously described (17). Bacterial cells. E. coli RRI (21) was used for the expression of the rho p21 protein. This strain carries the temperaturesensitive gene product cI ts (3). Protein expression and analysis. Bacterial cells containing the rho gene expression vector were grown in 1 liter of NZY (21) broth supplemented with 50 p.g of ampicillin per ml at 30°C. When an A590 of about 0.5 was reached, cells were transferred to 42°C and incubated at 250 rpm for 3 h. After centrifugation at 2,500 rpm for 10 min in a Sorvall RT6000 centrifuge at 4°C, pellets were washed twice in 100 ml of 50 mM Tris hydrochloride-5 mM EDTA-100 mM NaCl (pH 7.5) and sonicated for 60 s several times. After centrifugation at 12,000 rpm for 10 min in a Sorvall SS34 rotor, rho p21 proteins were solubilized in 25 ml of 7 M urea-20 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 7.0) and clarified at 30,000 rpm for 30 min in a Beckman ultracentrifuge. Supernatants were then collected and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in equal volumes of sample buffer as

described previously (18). Bacterial clones expressing the normal Harvey ras p21 protein or the mutant ras p21 protein (Lys-12) were processed in a similar manner, and both proteins were purified as described previously (18). For comparative studies, normal ras p21 was used unless otherwise indicated. Protein purification. Samples of 7 M urea extracts obtained as described above were subjected to further purification by chromatography through a Sephadex G-100 column (90 by 2.5 cm) with 7 M urea-20 mM MES (pH 7.0). Fractions of 3 ml were collected and analyzed by SDS-PAGE to estimate purity and by a GTP-binding assay (described below) to estimate the activity of rho p21. Fractions containing 90 to 95% purified rho p21 were pooled and dialyzed against 20 mM MES (pH 7.0)-10% glycerol, and the concentrations were estimated by the Bradford assay system (4). The usual purification protocol yielded concentrations of approximately 0.15 to 0.2 mg of rho p21 per ml at >90% purity. Specific activities were usually around 0.05 to 0.2 pmol of [a-32P]GTP bound per pmol of total protein. Samples were kept at -70°C for further characterization. GTP- and GDP-binding assays. GTP binding and GDP binding by rho and ras p21 proteins were assayed by incubation at various temperatures in 400 ,ul of GTP-binding buffer (20 mM Tris hydrochloride [pH 8.0], 0.5 mM MgCl2, 10 mM DTT, 100 ,ug of bovine serum albumin per ml) for different times in the presence of 1 ,uM [a-32P]GTP (3,000 Ci/mmol; ICN Pharmaceuticals Inc.) or [3H]GDP (10.8 Ci/ mmol; Amersham Corp.). Samples were passed through BA85 nitrocellulose filters (Schleicher & Schuell, Inc.), and the filters were washed twice with 5 ml of ice-cold GTP-

3621

binding buffer. After the filters were washed, the radioactivity of the GTP- or GDP-p21 complex retained by the filter was determined in a scintillation counter. Scatchard plot analysis of the highly purified rho p21 protein demonstrated single-site-binding kinetics for both GDP and GTP. Assuming that the molecular weight of the rho p21 protein is 21,000 and that 1 mol of nucleotide was bound per mol of protein, we estimated that 5 to 20% of the total p21 protein was active for GTP and GDP binding. GTPase assay. Approximately 10 pmol of purified protein was incubated at 37°C in 1.6 ml of GTPase buffer (50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES] [pH 8.0], 0.5 mM MgSO4, 100 mM NaCl, 0.25 mM 5'-adenyl-imido diphosphate, 5 mM DTT) containing 10 ,uM [y-32P]GTP (1,361 Ci/mmol; ICN). At various times, 100-jil aliquots from the reaction mixture were mixed with 500 ,ul of ice-cold 0.7 M perchloric acid containing 25 mM H2KPO4 and 4% activated charcoal. Samples were then vortexed, incubated on ice for 15 min, and centrifuged in a Microfuge, and the radioactivity contained in the supernatants was analyzed in a scintillation counter. To eliminate the actual GTP-binding activity for each protein, the same amounts of proteins were incubated at the same time under conditions similar to those described above, except that [a-32P]GTP (2,903 Ci/mmol; ICN) was used instead of [y-32P]GTP. Aliquots of 100 pul were analyzed for GTP binding as described above. GTPase values were then calculated by considering the actual values for the GTP-binding activity under the same experimental conditions. Both GTP-binding and GTPase assays were performed in triplicate.

RESULTS Expression of the A. californica rho gene in E. coli. A comparison of the sequence of the mammalian Harvey ras gene with that of the A. californica rho gene revealed the conservation of a convenient unique HindIII restriction site in both genes. This site is located at codons 5 to 6 of the ras gene and at codons 7 to 8 of the rho gene, with conservation of the respective reading frames. We have previously reported the construction of vectors which direct the expression of high levels of mammalian ras genes in E. coli (17, 18). One such vector, pJCL-41, produces the expression of the viral Harvey ras p21 protein, as described in detail elsewhere (18). To express the rho gene, we first removed the ras sequences from pJCL-41 as an 850-base-pair HindIII fragment and subcloned the resulting plasmid carrying the first six codons of the ras gene under the control of the regulatory sequences, as in pJCL-41 (data not shown). The newly generated plasmid was designated pJCL-1 (Fig. 1). The A. californica rho gene was originally cloned from a cDNA library in pSP6-2 as an EcoRI fragment (20). A clone which carried the rho gene in the orientation such that the complete coding sequence with the exception of the first six codons could be extracted as a 1-kilobase-pair HindIlI fragment was selected (Fig. 1). This fragment was then cloned into the Hindlll site of plasmid pJCL-1 in the proper orientation, producing the expression vector pRho-37. Digestion with a series of restriction enzymes, including AccI, NheI, RsaI, Sacl, NcoI, XmnI, NdeI, and EcoRI, demonstrated the identity of the 1-kilobase-pair fragment and its orientation. Restriction analysis indicated that plasmid pRho-37 indeed carried the rho gene and that it was placed in the proper orientation. The PL promoter of phage A is regulated by the temperature-sensitive cI repressor encoded by pRK248 cI ts (3).

3622

MOL. CELL. BIOL.

ANDERSON AND LACAL E o a

H

H A

II

Hlnd Ill

W | atg gca gcg ata cga aag aag cct gtt ata tac cgt cgc tat gct ttc ttc gaa caa tat met ala ala lie arg lys lys leu val ile E H /e

/

K

EH

ro

E H

A

Ligase E

~~~B \_o ~~A

B

IA

Hlnd Ill

A

p p

I ATG ACA GAA TAC AAG CTT GTG GTG r I IJITAC TGT CTT ATG TTC GAA CAC CAC LrJ A MET THR GLU TYR LYS LEU VAL VAL

ATG ACA GAA TAC Aag c t t gtt ata | h | TAC TGT CTT ATG TTC GAa caa tat MET THR GLU TYR lye leu val lie

FIG. 1. Generation of an expression plasmid for the rho p21 protein. A 1-kilobase-pair Hindlll fragment from plasmid pSP6-2 (20) carrying the entire A. californica rho p21-coding sequence except for the first six codons was purified from a 1% agarose gel. This fragment was introduced in the appropriate orientation into the HindlIl site of plasmid pJCL-1, generated by removal of the 850-base-pair HindIII fragment of the Harvey ras p21 gene from plasmid pJCL-41 (17). The resulting new plasmid, pRho-37, carries the A. californica rho gene under the control of the PL promoter of X phage (PL), followed by a consensus Shine-Dalgarno sequence (ribosome-binding site) and the first four codons from the v-baslras gene provided by plasmid pJCL-41 (17). Plasmid pRho-37 carries a chimeric gene in which the first four codons of the ras gene are fused to A. californica rho gene codons 7 to 192, followed by the TGA stop codon of the rho gene. The resulting gene product, designated the rho p21 protein, has 190 amino acids. Restriction enzyme abbreviations: A, AccI; B, BamHI; E, EcoRI; H, Hindlll; P, PstI.

When cells carrying the pRK248 cI ts plasmid and PLcontrolled vector are grown at 30°C, the cI product represses the expression of the heterologous genes. Expression can be achieved by inactivation of cI function when cells are incubated at 42°C. Figure 2 shows the results obtained when E. coli strains carrying both pRho-37 and pRK248 cI ts were grown at 30°C and then shifted to 42°C. The E. coli strains carrying the pJCL-1 and pRK248 cI ts plasmids are shown, as is a negative control. A protein with an apparent molecular weight of approximately 21,000 was readily observed specifically in the pRho-37-containing clone when incubated at 42°C. Maximal expression levels were achieved when cells were grown at 30°C until an A590 of about 0.5 was reached and then transferred to 42°C for 3 h (results not shown). Under optimal conditions, the level of expression of the rho p21 protein was estimated to be approximately 5% of the total protein content by densitometric scanning of Coomassie blue-stained gels similar to that shown in Fig. 2. Purification of rho p21. The rho p21 protein expressed in E. coli was found as a >90% insoluble product after cell disruption (data not shown). Thus, to solubilize the protein for further purification, we utilized a protocol similar to that previously described for the purification of biologically active ras p21 proteins from bacterial extracts (18). Cells were grown at 30°C and transferred to 42°C as described in Materials and Methods. After lysis, bacterial extracts were solubilized in 20 mM MES-7 M urea (pH 7.0) and resolved on a Sephadex G-100 column with the same buffer. Column fractions were collected and analyzed for GTP-binding activity and by SDS-PAGE. A peak of activity which coeluted with the rho p21-specific band was detected (Fig. 3). Frac-

tions were pooled, extensively dialyzed against 20 mM MES (pH 7.0)-10% glycerol, and stored at -70°C for further analysis. When bacterial extracts carrying the pJCL-1 and pRK248 cI ts plasmids were processed under similar conditions as were those carrying the pRho-37 plasmid, no detectable GTP-binding activity was found within the entire gradient (data not shown). Thus, these results demonstrated that the rho p21 protein was a GTP-binding protein. Densitometric analysis of several SDS-PAGE gels of the Sephadex G-100 fractions indicated that the rho p21 protein was between 80 and 90% pure. Specific GTP activities were approximately 0.05 to 0.2 pmol of GTP bound per pmol of total protein, as determined by the Bradford method (4). These results were similar to those previously reported for ras p21 proteins expressed and purified under similar conditions (18). GTP-binding requirements. Analysis of the guanine nucleotide-binding properties of the rho p21 protein was carried out by the standard filter assay procedure as previously described (18). Conditions for optimal binding were determined by selecting individual optima for known factors which affect the binding properties of other well-characterized G proteins. GTP binding to the rho p21 protein was in some cases compared to GTP binding to the bacterially expressed and purified ras p21 protein. The rho p21 protein exhibited optimal binding activity at pH 7 to pH 8.5, while the ras p21 protein exhibited optimal binding activity at about pH 8 to pH 9 (Fig. 4). Both proteins were rapidly inactivated at pHs lower than 6.5, following almost identical inactivation patterns. The requirement of sulfhydryl compounds and Mg2+ ions

VOL. 7, 1987


pJCL-1 300 420

MW

pRho-37 30° 420

200K -+

92K-' 68K

-

£

-

43K

25.7K

-.9

3623

Competition for guanine nucleotide binding by rho p21. The binding of either GDP or GTP to known G proteins is highly specific and is not displaced by competition with other nucleotides (25, 26). Thus, we investigated the effects of a 100-fold molar excess of other nucleotides on GTP binding by the rho and ras p21 proteins. GTP binding by the ras p21 protein was not efficiently competed for (around 10% or less inhibition) by ATP, CTP, TTP, UTP, guanine, guanosine, guanosine-2'-phosphate, guanosine-3'-phosphate, guanosine-2',3'-cyclic phosphate, or dibutyryl-cyclic GMP (Table 1). Partial inhibition (40 to 60%) was observed with pGp, dGMP, pGpp, and guanosine-diphosphoglucose. A substantial reduction (>70% competition) was observed with pppppG, ppppG, ppGpp, GTP, GDP, ppGp, and guanosine 5'-O-(3-thio-triphosphate). In contrast to these results, significant differences were found in the competition for GTP binding by the rho p21 protein with several compounds. The most striking differences were found with ATP, CTP, TTP,

-rho 18.4K

ri9i

12.3KFIG. 2. Expression of the rho p21 protein in E. coli. Bacterial cultures carrying plasmid pJCL-1 or pRho-37 were grown at 30°C until an A590 of about 0.5 was reached. Aliquots were further incubated at either 30 or 42°C, and cells were treated as described in Materials and Methods. Samples from each culture were analyzed by SDS-PAGE with 12.5% polyacrylamide gels and Coomassie blue stained by standard protocols. Molecular weight (MW) standards were as follows: myosin (200,000 [200K]); phosphorylase b (92K); bovine serum albumin (68K); ovalbumin (43K); a-chymotrypsin (25.7K); ,B-lactoglobulin (18.4K); and cytochrome c (12.3K).

for the binding properties of G proteins has been previously demonstrated (22). The requirement of either DTT or 1mercaptoethanol (ME) for rho and ras p21 proteins were compared. The optimal GTP-binding activity of rho p21 required approximately 10-fold-higher concentrations of either DTT or ME than did that of ras p21 (Fig. 5). The presence of Mg2+ was required but was not absolute, since there was some specific GTP binding in the absence of Mg2+ and in the presence of EDTA (Fig. 6). Scatchard analysis of GDP and GTP binding. The affinity of guanine nucleotides for proteins can be accurately measured by Scatchard analysis with the filter assay technique (9, 18, 22). To establish the rho p21 dissociation constants (Kds) for both GDP and GTP, we first established the temperature dependency of nucleotide binding. Maximum binding was achieved for either nucleotide at 37°C in about 30 min but binding was extremely poor at 0°C (Fig. 7). This result was in agreement with previous results obtained with the bacterially expressed ras p21 protein (18). Kinetics similar to those obtained at 37°C were observed at 24°C but at a slightly slower rate. Scatchard analysis was then performed at 37°C as a function of GTP concentration (Fig. 8). Saturation was reached at about 0.6 ,uM (Fig. 8A). The results obtained were consistent with single-site-binding kinetics (Fig. 8B), with an estimated Kd of 3.03 x 10-7 M. A similar analysis was carried out as a function of GDP concentration (Fig. 9). Saturation was reached at 0.4 ,uM (Fig. 9A), and the estimated Kd was 7.6 x 10-8 M. From these results it appears that the rho p21 protein has an approximately fourfoldhigher affinity for GDP than for GTP.

FRACTION

A MW

St S 60 64O66 7276 n0 6462 96

100 112

I'..'I

200K96K68K -

46K-

26K

18.4K

-

a

12.3K-

E

CL

-

c-

._

c

200000

cL

ca

0

50

70

90 110 FRACTION FIG. 3. Purification of the rho p21 protein expressed in E. coli. Bacterial cultures were incubated at 30°C and then shifted to 42°C as described in Materials and Methods. Cells were collected and processed to solubilize rho p21 as described in Materials and Methods. Solubilized material was applied to a Sephadex G-100 column, and aliquots of 10 F±l from each fraction were analyzed by polyacrylamide gel electrophoresis (A) or for GTP-binding activity by the filter assay method at 37°C for 60 min (B). Molecular weight markers are the same as those described in the legend to Fig. 2.

MOL. CELL. BIOL.

ANDERSON AND LACAL

3624

ras p21 protein for GDP and GTP, measured under similar

-_ rho

3.0-

conditions, were about 10-fold lower than those of the rho p21 protein, these results might be a reflection of the lower affinity of rho p21. However, the ability of ATP, CTP, TTP,

-- ras

and UTP to compete for GTP binding might be a consequence of the lower specificity in the binding pattern of rho CL p21. IU |/GTPase activity of rho p21. GTPase activity is a common property of regulatory guanine nucleotide-binding proteins. t0 1.0 This property constitutes the basis for the activation | and inactivation cycle of G proteins, establishing the mechanism of regulation of their function (for reviews, see references 11, 23, and 30). To strengthen the conclusion that the 0.0 4 46 rho p21 protein functions as a real G protein, we investigated 8 10 11 22 11 44 10 its GTPase activity and compared it with that of two different ras p21 proteins. The rho p21 protein had an associated pH GTPase activity of around 0.1 mol/min per mol of p21, an activity which was almost identical to that of the normal ras FIG. 4. GTP binding to rho and ras p21 proteins at different pHs. p21 protein and similar to that of other well-characterized G Purified ras and rho p21 proteins (1 1lg) were incubated in 400 Al of * * GTP-binding buffer adjusted to the indicated pH and containing 1 B (Fig. 10). By contrast, a mutant ras p21 protein with ,uM [a-32P]GTP (1.5 x 105 cpm/pmol). Binding was established by proteLns a Gly-to-Lys substitution at position 12 had a decreased the filtration assay. Results are the means of three independent experiments, with a variation of less than 10%o from experiment to GTPase activity of around 0.025 mol/min per mol of p21, in agreement with previous findings (18). Thus, the rho p21 experiment, protein had a GTP hydrolytic activity comparable to that of known regulatory GTP-binding proteins. and UTP, with 50 to 60% inhibition, and with ppGp and DISCUSSION guanosine 5'-O-(3-thio-triphosphate), both with more than 90% inhibition. Under similar conditions, GDP and GTP We have constructed an expression vector carrying the A. were also better competitors for GTP binding by rho p21 californica rho gene under the control of the bacteriophage X than for that by ras p21. Since the affinity constants of the PL promoter to produce large amounts of the rho p21 protein E 2.0

-

30000 0

c

._

20000

3

._

:3

0

._

O

10000

0 0

2

4

6

8

10

DTT, mM

ME, mM

a

c

5

0

._c

C

C.

3

a

a,

-r,

1.0 1.2 0.6 0.8 0.2 0.4 0.0 4 5 6 3 DTT, mM ME, mM FIG. 5. Sulfhydryl compound requirement for GTP binding to rho and ras p21 proteins. Purified rho and ras p21 proteins (1 pLg) were incubated in 0.4 ml of 20 mM Tris hydrochloride (pH 8.0)-0.5 mM MgCl2-100 ,ug of bovine serum albumin per ml containing 1 ,M [ct-32P]GTP (1.02 x 104 cpm/pmol) for 60 min at 37°C and filtered as described in Materials and Methods. Different amounts of DTT or ME were included in the reaction mixtures. Results are the means of two independent experiments. 0

1

2

VOL. 7, 1987


80000

E 0.

60000

:a

40000

CL Qa.

20000 0

0.0

0.4

0.8

1.2

1.6

2.0

Mg2+, mM FIG. 6. Mg2+ ion requirement for GTP binding to rho and ras p21 proteins. Bacterially expressed and purified rho and ras p21 proteins (2 Lg) were incubated in 0.4 ml of 30 mM Tris hydrochloride (pH 8.0)-10 mM DTT-0.5 mM EDTA-100 p.g of bovine serum albumin per ml containing 1 ,uM [a-32P]GTP (2.5 x 104 cpm/pmol) for 60 min at 37°C. Different concentrations of MgCl2 were added to the reaction mixtures, and incubation and processing of the samples were done as described in Materials and Methods. Results are the means of two independent experiments.

in E. coli. The expression vector was generated by in vitro recombination of the amino-terminal portion of the mammalian Harvey ras gene and the A. californica rho gene. The resulting product was a 190-amino-acid polypeptide which has the entire amino acid sequence of the rho p21 protein with the first six amino acids replaced by the first four amino acids from the ras p21 protein. Since the different members of the ras family from S. cerevisiae show a certain degree of heterogeneity at the amino-terminal region with no apparent effect on the in vitro or in vivo activities of the ras p21 protein, we reasoned that the minor modification introduced in the rho p21 protein would likely have little or no effect on its biochemical properties. We have purified the rho p21 protein to approximately 90% purity by a procedure similar to that previously used by us for the purification of biologically active ras p21 proteins expressed in E. coli (15, 17, 18). Both the biochemical and

3625

TABLE 1. Competition for GTP binding by rho p21a Binding (% of control) of: Compound ras p21 rho p21 None 100 100 GTP 15 4 GDP 28 4 ATP 85 38 CTP 94 48 TTP 91 47 UTP 89 38 Diphosphoguanosine 5'-O-(3-thio-triphosphate) 30 5 Guanine 100 90 Guanosine 100 93 Guanosine-2'-phosphate 100 94 Guanosine-3'-phosphate 100 90 Guanosine-2',3'-cyclic phosphate 112 130 dGMP 52 57 pGp 60 44 pGpp 48 48 ppGpp 4 5 ppppG 4 5 ppGp 28 5 pppppG 2 4 Dibutyryl-cyclic GMP 85 77 24 Guanosine-diphosphoglucose 40 a Purified rho p21 (1 Fig) was incubated for 60 min at 37C in GTP-binding buffer containing 1 FiM [a_-3P]GTP in the absence or presence of 100 FM concentrations of the indicated compounds. After incubation, samples were processed as described in Materials and Methods. Results are the means of two independent experiments, with 100lo of controls being 3.0 x 105 to 3.9 x 10- cpm (ras p21) or 1.3 x 10' to 1.6 x 10' cpm (rho p21).

biological properties of the ras p21 proteins are conserved after purification, suggesting that a similar purification protocol for the rho p21 protein would allow for its characterization. A comparison of the amino acid sequences of the encoded products of the mammalian Harvey ras and A. californica rho genes has revealed 35% homology (20). This finding suggests that both genes have arisen from a common ancestral gene. Conservation of sequences is clustered in a small number of regions at positions 5 to 21, 37 to 64, 109 to 120,

Z

.5

E

E

C

3Ca :5

CL

a.

c,

a

100

TIME (min) TIME (min) FIG. 7. Temperature dependency of GTP and GDP binding to the rho p21 protein. Purified rho p21 protein was incubated at 0, 24, or 37°C in GTP-binding buffer containing 1 ,uM [a-32P]GTP (2.7 x 104 cpm/pmol) or [3H]GDP (9,500 cpm/pmol). Incubation at the indicated temperatures was done for different periods of time, and samples were processed as described in Materials and Methods. Results are the means of three independent experiments.

3626

ANDERSON AND LACAL

ML CELL. EL BIOL. MOL. IL 4

3

0

E

0.

I

2

.0

0.

C,

1

0 0

200

400

600

800 1000 1200 1400

GTP, nM

GTP-bound (pmol)

FIG. 8. Scatchard analysis of GTP binding to the purified rho p21 protein. Bacterially purified rho p21 protein (1 pLg) was incubated in 0.4 ml of GTP-binding buffer containing different concentrations (5 x 10-9 to 1.5 x 10-6 M) of [a-_32P]GTP (4.3 x 106 CpM/pMol) for 60 min at 370C. Samples were filtered as described in Materials and Methods. (A) GTP binding as a function of GTP concentration. (B) Scatchard plot.

and 141 to 170 (from Harvey ras p21). Several reports have indicated that at least these four regions are involved in the GTP-binding function of ras p21 proteins (5, 8, 12, 16, 28, 32). However, the presence of large divergent regions suggests that members of the two families might interact with different effector molecules and, in so doing, perform dif-

ferent functions. One of the best known activities of the ras p21 proteins is their ability to bind GTP and GDP. We demonstrated that the rho p21 protein shares this property but with some differences in its characteristics. Optimal binding by rho p21 occurred at around pH 7 to pH 8.5, with a complete and rapid inactivation at pHs lower than 6.5. By contrast, ras p21 was more resistant to alkaline treatment but showed similar inactivation kinetics at pHs lower than 6.5. A major difference was found at the level of the sulfhydryl compound requirement. To achieve full binding activity, the rho p21 protein required 5- to 10-fold-higher concentrations of either DTT or ME. Whether differences in the requirement for sulfhydryl compounds reflect major structural differences between the proteins is not known. The fact that only one of the four highly conserved cysteines among mammalian ras p21 proteins was found in the rho p21 protein suggests different structures for both proteins. This hypothesis is

supported by evidence for the existence of a disulfide bridge in ras proteins (13, 29). Thus, a possible explanation for the difference in the requirement of sulthydryl compounds might be that the rho p21 protein acquires a different conformation than the ras p21 protein after purification and that renaturation is required for full binding activity. One of the most important differences in the binding activities of rho p21 and ras p21 was an almost 10-fold difference in the dissociation constants (Kds) estimated by Scatchard analysis. Kds of 1.02 x 10-8 and 3.74 x 10-8 M were previously reported for GTP and GI]iP, respectively, for the normal ras p21 protein (18). The rho p21 protein had Kds of 3.03 X 10-7 and 7.6 x 10-8 M for GTP and GDP, respectively, indicating a 5- to 10-fold-lower affinity of rho p21 than of ras p21. These results are in agreement with those of competition experiments with other nucleotides. Moreover, a 100-fold excess of nonguanine nucleotides competed more efficiently for GTP binding to the rho p21 protein than to the ras p21 protein. Similarly, guanosine nucleotides competed more efficiently for GTP binding to rho p21 than to ras p21. These results imply that rho p21 had a lower specificity for GTP and GDP binding, consistent with different functions for rho and ras p21 proteins. One alternative explanation could be that the rho p21 protein is more 2.5,

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.0

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GDP-bound (pmol) FIG. 9. Scatchard analysis of GDP binding to the purified rho p21 protein. Bacterially purified rho p21 protein (1 ~i.g) was incubated in 0.4 ml of GTP-binding buffer containing different concentrations (10 x 10-9 to 3 x 10-6 M) of [3H]GDP (11,970 cpmlpmol) for 60 min at 370C. Samples were filtered as described in Materials and Methods. (A) GDP binding as a function of GDP concentration. (B) Scatchard plot.


VOL. 7, 1987 15I

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TIME (min) FIG. 10. GTPase activity of the purified rho and ras p21 proteins. Bacterially expressed rho and ras p21 proteins were purified as described in the text. Equivalent amounts of each protein (-10 pmol), based on their ability to bind GTP, were diluted in GTPase buffer and analyzed as described in Materials and Methods. Symbols: A, rho p2l; O, normal ras p2l; *, mutant ras p21 (Lys-12)

10.

11. 12.

(18).

sensitive to the purification process, so that full binding activity as well as specificity may have been altered. However, we believe that this is not the case, since the ability of both the normal ras p21 protein and the rho p21 protein to hydrolyze GTP is almost identical. Nonetheless, our findings that the rho p21 protein binds guanine nucleotides with high affinity strongly argue that it is a G protein. One of the most important features of G proteins with known regulatory functions is their ability to slowly hydrolyze bound GTP at a rate of about 0.010 to 0.100 mol/min per mol of protein. The active complex (G protein-GTP) becomes inactivated by this mechanism and remains inactive while the protein is associated with GDP, until the replacement of GDP with GTP occurs (reviewed in references 11, 23, and 30). The GTPase activity and guanine nucleotide interchange of these proteins are regulated by specific mechanisms (11, 23, 30). Thus, our findings that the bacterially purified rho p21 protein efficiently binds both GDP and GTP and has a GTPase activity comparable to that of well-known G proteins strengthen our conclusion that the rho p21 protein may function as a regulatory protein. The availability of large amounts of the rho p2u protein will help in the understanding of its function. ACKNOWLEDGMENTS We thank R. Axel for kindly providing the cDNA clone of the A. californica rho gene and S. Aaronson for his support and critical reading of the manuscript. LITERATURE CITED 1. Ahnn, J., P. E. March, H. E. Takfrl,and M. Inouye. 1986. A GTP-binding protein of Escherichia coli has homology to yeast RAS proteins. Proc. Natl. Acad. Sci. USA 83:8849-8853. 2. Beckner, S. K., S. Hattori, and T. Y. Shih. 1985. The ras oncogene productp2u is not a regulatory component of adenylate cyclase. Nature (London) 317:71-72. 3. Bemard, H., and D. R. Heosinka. 1979. Use of the phage

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